PROJECT SUMMARY Cost-effective on-site assay technologies for pathogen detection are sorely lacking, even though they are essential in solving many global challenges such as food and water borne infections and disease outbreaks. For example, each year foodborne diseases from E. coli and salmonella contamination alone cost $6.4 billion in the US. One of the most common strategies for foodborne pathogen detection is laboratory-based polymerase chain reaction (PCR) of a single pathogen type that takes about 24 h followed by confirmation using culture-based methods that take a minimum of 1 week. Additionally, a majority of techniques reli only on DNA whereas RNA is a better indicator of viable bacteria. Ideal technology should target detection of multiple pathogens through the detection of specific nucleic acid in a portable on-site detection platform. However, the detection of multiple nucleic acid targets in a single assay is still problematic, and transitioning a robust, multiplexed nucleic acid detection assay into a low-cost, rapid, on-site detection device is a significant challenge. To that end, our overall goal is to develop a flow-strip multiplex pathogen detection platform that will fulfill the following criteria: simple sample preparation, rapid multiplexed DNA/RNA target amplification and incorporation of reporter in a single pot, portable platform with colorimetric detection for on-site and inexpensive monitoring. Here, we propose to use recombinase polymerase amplification (RPA) and reverse transcriptase-RPA to incorporate unique zinc-finger protein (ZFP) binding motifs (referred to as ?Ztags?) and reporter moieties into the amplification product from nucleic acid targets of interest in order to detect the presence of viable pathogen. The amplified product will be captured using immobilized ZFPs via specific ZFP- Ztag interaction for real-time reporter-based detection on a flow-strip platform. Our preliminary data demonstrated that we can incorporate both Ztag and reporter into the target nucleic acid using the RPA method and detect as low as 10 copies of target. Additionally, the proposed method does not need any extraction step as our preliminary data indicates that the RPA method is compatible with the cell lysis reagent. We plan to achieve our goal by pursuing the following specific aims, namely, (1)(a) Design and optimization of the amplification as well as Ztag and reporter incorporation using single-step, one-pot RPA for the detection of the pathogenic strains E. coli O157, E. coli O26, and E. coli O121. (b) Evaluation of the binding affinity between ZFP and target-incorporated Ztag; (2) Design and development of a flow-strip-based, multiplex DNA/RNA detection platform for the different E. coli strains; (3)(a) Characterization and validation of the complete assay using water and food samples. (b) Evaluation of the long-term stability of the flow strip platform. The proposed research is significant because it will provide a simple and inexpensive multiplex pathogen detection platform that could achieve a global impact due to reducing the economic burden of food and water borne diseases in the developed world and improving access to detection technologies in low- resource environments.